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Inhibition of porcine reproductive and respiratory syndrome virus infection by recombinant adenovirus- and/or exosome-delivered the artificial microRNAs targeting sialoadhesin and CD163 receptors.

Zhu L, Song H, Zhang X, Xia X, Sun H - Virol. J. (2014)

Bottom Line: Both PRRSV ORF7 copy number and viral titer were reduced significantly by transduction of PAMs with the two rAds and/or by treatment with the two amiRNA-containing exosomes.The additive anti-PRRSV effect between the two amiRNAs was relatively long-lasting (96 h) and effective against three different viral strains.These results suggested that Sn- and CD163-targeted amiRNAs had an additive anti-PRRSV effect against different viral strains.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, China. 1029765408@qq.com.

ABSTRACT

Background: The current vaccines failed to provide substantial protection against porcine reproductive and respiratory syndrome (PRRS) and the new vaccine development faces great challenges. Sialoadhesin (Sn) and CD163 are the two key receptors for PRRS virus (PRRSV) infection of porcine alveolar macrophages (PAMs), but the artificial microRNA (amiRNA) strategy targeting two viral receptors has not been described.

Methods: The candidate miRNAs targeting Sn or CD163 receptor were predicted using a web-based miRNA design tool and validated by transfection of cells with each amiRNA expression vector plus the reporter vector. The amiRNA-expressing recombinant adenoviruses (rAds) were generated using AdEasy Adenoviral Vector System. The rAd transduction efficiencies for pig cells were measured by flow cytometry and fluorescent microscopy. The expression and exosome-mediated secretion of amiRNAs were detected by RT-PCR. The knock-down of Sn or CD163 receptor by rAd- and/or exosome-delivered amiRNA was detected by quantitative RT-PCR and flow cytometry. The additive anti-PRRSV effect between the two amiRNAs was detected by quantitative RT-PCR and viral titration.

Results: All 18 amiRNAs validated were effective against Sn or CD163 receptor mRNA expression. Two rAds expressing Sn- or CD163-targeted amiRNA were generated for further study. The maximal rAd transduction efficiency was 62% for PAMs at MOI 800 or 100% for PK-15 cells at MOI 100. The sequence-specific amiRNAs were expressed efficiently in and secreted from the rAd-transduced cells via exosomes. The expression of Sn and CD163 receptors was inhibited significantly by rAd transduction and/or amiRNA-containing exosome treatment at mRNA and protein levels. Both PRRSV ORF7 copy number and viral titer were reduced significantly by transduction of PAMs with the two rAds and/or by treatment with the two amiRNA-containing exosomes. The additive anti-PRRSV effect between the two amiRNAs was relatively long-lasting (96 h) and effective against three different viral strains.

Conclusion: These results suggested that Sn- and CD163-targeted amiRNAs had an additive anti-PRRSV effect against different viral strains. Our findings provide new evidence supporting the hypothesis that exosomes can also serve as an efficient small RNA transfer vehicle for pig cells.

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Validation of Sn and CD163 receptor-targeted amiRNAs. (A) The schematic structure of amiRNA expression vector. PCMV, immediate early promoter of cytomegalovirus; miR, miR-flanking sequences of mouse BIC non-coding mRNA; Pre-amiRNA, double–stranded oligonucleotide for amiRNA; TK pA, TK gene poly(A) signal of human simplex herpes virus. (B) The schematic structure of the reporter vectors for amiRNA validation. PCMV, immediate early promoter of cytomegalovirus; Sn/CD163, porcine Sn or CD163 receptor cDNA; GFP, green fluorescent protein coding sequence; SV40 pA, poly(A) signal of SV40 virus. (C or D) NIH 3 T3 cells were transfected with different amiRNA expression vector plus the reporter vector, and the GFP-positive cell numbers were measured by flow cytometry 24 h after transfection. The know-down efficiency of each amiRNA was expressed as the percent inhibition of total fluorescence in the cell culture co-transfected with the amiRNA expression vector and the reporter vector.
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Fig1: Validation of Sn and CD163 receptor-targeted amiRNAs. (A) The schematic structure of amiRNA expression vector. PCMV, immediate early promoter of cytomegalovirus; miR, miR-flanking sequences of mouse BIC non-coding mRNA; Pre-amiRNA, double–stranded oligonucleotide for amiRNA; TK pA, TK gene poly(A) signal of human simplex herpes virus. (B) The schematic structure of the reporter vectors for amiRNA validation. PCMV, immediate early promoter of cytomegalovirus; Sn/CD163, porcine Sn or CD163 receptor cDNA; GFP, green fluorescent protein coding sequence; SV40 pA, poly(A) signal of SV40 virus. (C or D) NIH 3 T3 cells were transfected with different amiRNA expression vector plus the reporter vector, and the GFP-positive cell numbers were measured by flow cytometry 24 h after transfection. The know-down efficiency of each amiRNA was expressed as the percent inhibition of total fluorescence in the cell culture co-transfected with the amiRNA expression vector and the reporter vector.

Mentions: Computational prediction represents an effective strategy for identification of the candidate siRNAs or miRNAs that can be validated experimentally. We analyzed the first 1751-nt sequence of porcine Sn mRNA and the 1511-nt sequence of porcine CD163 mRNA for candidate miRNAs. Ten top-scoring miRNA sequences for each target were reported and 18 of them (Table 1) were selected for amiRNA vector construction (Figure 1A). To facilitate the candidate miRNA validation, the first 1751-bp sequence of the Sn cDNA or the 1511-bp sequence of the CD163 cDNA was cloned in frame with the green fluorescent protein (GFP) coding sequence in pEGFP-N1 vector to produce the reporter vector pSn-GFP or pCD163-GFP (Figure 1B). NIH 3 T3 cells were co-transfected with each amiRNA expression vector and the reporter vector, and the cell culture was assayed for GFP+ cells by flow cytometry. By using pSn-GFP-transfected cells as the reference, transfection with each amiRSn expression vector plus the reporter vector resulted in GFP+ cell number reductions ranging from 39.6% to 96.3% (Figure 1C). Similarly, transfection with each amiRCD163 expression vector plus the reporter vector led to GFP+ cell number reductions ranging from 53.3% to 88.5% (Figure 1D). The most effective amiRSn-2 and amiRCD163-2, as well as an irrelevant amiRcon (Table 1), were selected for further study.Table 1


Inhibition of porcine reproductive and respiratory syndrome virus infection by recombinant adenovirus- and/or exosome-delivered the artificial microRNAs targeting sialoadhesin and CD163 receptors.

Zhu L, Song H, Zhang X, Xia X, Sun H - Virol. J. (2014)

Validation of Sn and CD163 receptor-targeted amiRNAs. (A) The schematic structure of amiRNA expression vector. PCMV, immediate early promoter of cytomegalovirus; miR, miR-flanking sequences of mouse BIC non-coding mRNA; Pre-amiRNA, double–stranded oligonucleotide for amiRNA; TK pA, TK gene poly(A) signal of human simplex herpes virus. (B) The schematic structure of the reporter vectors for amiRNA validation. PCMV, immediate early promoter of cytomegalovirus; Sn/CD163, porcine Sn or CD163 receptor cDNA; GFP, green fluorescent protein coding sequence; SV40 pA, poly(A) signal of SV40 virus. (C or D) NIH 3 T3 cells were transfected with different amiRNA expression vector plus the reporter vector, and the GFP-positive cell numbers were measured by flow cytometry 24 h after transfection. The know-down efficiency of each amiRNA was expressed as the percent inhibition of total fluorescence in the cell culture co-transfected with the amiRNA expression vector and the reporter vector.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig1: Validation of Sn and CD163 receptor-targeted amiRNAs. (A) The schematic structure of amiRNA expression vector. PCMV, immediate early promoter of cytomegalovirus; miR, miR-flanking sequences of mouse BIC non-coding mRNA; Pre-amiRNA, double–stranded oligonucleotide for amiRNA; TK pA, TK gene poly(A) signal of human simplex herpes virus. (B) The schematic structure of the reporter vectors for amiRNA validation. PCMV, immediate early promoter of cytomegalovirus; Sn/CD163, porcine Sn or CD163 receptor cDNA; GFP, green fluorescent protein coding sequence; SV40 pA, poly(A) signal of SV40 virus. (C or D) NIH 3 T3 cells were transfected with different amiRNA expression vector plus the reporter vector, and the GFP-positive cell numbers were measured by flow cytometry 24 h after transfection. The know-down efficiency of each amiRNA was expressed as the percent inhibition of total fluorescence in the cell culture co-transfected with the amiRNA expression vector and the reporter vector.
Mentions: Computational prediction represents an effective strategy for identification of the candidate siRNAs or miRNAs that can be validated experimentally. We analyzed the first 1751-nt sequence of porcine Sn mRNA and the 1511-nt sequence of porcine CD163 mRNA for candidate miRNAs. Ten top-scoring miRNA sequences for each target were reported and 18 of them (Table 1) were selected for amiRNA vector construction (Figure 1A). To facilitate the candidate miRNA validation, the first 1751-bp sequence of the Sn cDNA or the 1511-bp sequence of the CD163 cDNA was cloned in frame with the green fluorescent protein (GFP) coding sequence in pEGFP-N1 vector to produce the reporter vector pSn-GFP or pCD163-GFP (Figure 1B). NIH 3 T3 cells were co-transfected with each amiRNA expression vector and the reporter vector, and the cell culture was assayed for GFP+ cells by flow cytometry. By using pSn-GFP-transfected cells as the reference, transfection with each amiRSn expression vector plus the reporter vector resulted in GFP+ cell number reductions ranging from 39.6% to 96.3% (Figure 1C). Similarly, transfection with each amiRCD163 expression vector plus the reporter vector led to GFP+ cell number reductions ranging from 53.3% to 88.5% (Figure 1D). The most effective amiRSn-2 and amiRCD163-2, as well as an irrelevant amiRcon (Table 1), were selected for further study.Table 1

Bottom Line: Both PRRSV ORF7 copy number and viral titer were reduced significantly by transduction of PAMs with the two rAds and/or by treatment with the two amiRNA-containing exosomes.The additive anti-PRRSV effect between the two amiRNAs was relatively long-lasting (96 h) and effective against three different viral strains.These results suggested that Sn- and CD163-targeted amiRNAs had an additive anti-PRRSV effect against different viral strains.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, China. 1029765408@qq.com.

ABSTRACT

Background: The current vaccines failed to provide substantial protection against porcine reproductive and respiratory syndrome (PRRS) and the new vaccine development faces great challenges. Sialoadhesin (Sn) and CD163 are the two key receptors for PRRS virus (PRRSV) infection of porcine alveolar macrophages (PAMs), but the artificial microRNA (amiRNA) strategy targeting two viral receptors has not been described.

Methods: The candidate miRNAs targeting Sn or CD163 receptor were predicted using a web-based miRNA design tool and validated by transfection of cells with each amiRNA expression vector plus the reporter vector. The amiRNA-expressing recombinant adenoviruses (rAds) were generated using AdEasy Adenoviral Vector System. The rAd transduction efficiencies for pig cells were measured by flow cytometry and fluorescent microscopy. The expression and exosome-mediated secretion of amiRNAs were detected by RT-PCR. The knock-down of Sn or CD163 receptor by rAd- and/or exosome-delivered amiRNA was detected by quantitative RT-PCR and flow cytometry. The additive anti-PRRSV effect between the two amiRNAs was detected by quantitative RT-PCR and viral titration.

Results: All 18 amiRNAs validated were effective against Sn or CD163 receptor mRNA expression. Two rAds expressing Sn- or CD163-targeted amiRNA were generated for further study. The maximal rAd transduction efficiency was 62% for PAMs at MOI 800 or 100% for PK-15 cells at MOI 100. The sequence-specific amiRNAs were expressed efficiently in and secreted from the rAd-transduced cells via exosomes. The expression of Sn and CD163 receptors was inhibited significantly by rAd transduction and/or amiRNA-containing exosome treatment at mRNA and protein levels. Both PRRSV ORF7 copy number and viral titer were reduced significantly by transduction of PAMs with the two rAds and/or by treatment with the two amiRNA-containing exosomes. The additive anti-PRRSV effect between the two amiRNAs was relatively long-lasting (96 h) and effective against three different viral strains.

Conclusion: These results suggested that Sn- and CD163-targeted amiRNAs had an additive anti-PRRSV effect against different viral strains. Our findings provide new evidence supporting the hypothesis that exosomes can also serve as an efficient small RNA transfer vehicle for pig cells.

Show MeSH
Related in: MedlinePlus